ANALYTICAL BIOCHEMISTRY (ABIC 301)
PRACTICAL 1-3: UV/Vis SPECTROSCOPY
DUE DATE: 21 AUGUST 2018
NOXOLO Z MABUYAKHULU : 21521506
SIHLE MHLONGO : 21707326
AYANDA KHANYE : 21615620
THABISO S ZUMA : 21707334
SILINDOKUHLE SHABANGU : 21612869
LUBISI SEBENZILE : 21641123
TITTLE: the use of ultraviolet visible (UV/Vis) spectroscopy to
determine absorbance and standard curve of nickel sulphate,
sugars and plants.
The ultraviolet-visible (UV/Vis) spectrometer is an instrument commonly used in laboratory
that analyses compounds in the ultraviolet and visible regions of the electromagnetic
spectrum. Unlike infrared spectroscopy (which looks at vibrational motion), ultraviolet-visible
spectroscopy looks at electronic transitions. It allows one to determine the wavelength and
maximum absorbance of compounds. From the absorbance information and using a
relationship known as Beer’s Law, is able to determine either the concentration of a sample
in this case three samples were observed nickel sulphate, sugar and plants if the molar
extinction coefficients are specific to particular compounds, therefore UV-Vis spectroscopy
will aid one in determining an unknown compound’s identity. (R Hogg and J Ledolter 2007).
Future improvements in UV-Vis spectrophotometers will focus on ease-of-use, portability,
and application specific instruments. UV-Vis analysis of solid samples and materials continues
to grow in areas such as solar cell research, semiconductors products, and coating materials.
Advances in light sources will provide new developments in conventional
spectrophotometers and handheld UV-Vis instrument. Further development in remote
sensors will enable more types of samples to be measured outside the laboratory. (A
The direct spectrophotometric determination of metal ions in multicomponent system is
often complicated by interferences from the formulation matrix and spectral overlapping.
Such interferences have been treated in many ways, such as using absorbance ratios at certain
wavelengths however, during the application methods the presence of spectral interferences
or spectral overlap would certainly lead to erroneous results. (H Akers 2003).
Spectrophotometry is an analytical technique of great utility for extracting both qualitative
and quantitative information from spectra composed of unresolved bands, and for
eliminating the effect of baseline shifts and baseline tilts. It consists of calculating and plotting
one of the mathematical derivatives of a spectral curve. Thus, the information content of a
spectrum is presented in a potentially more useful form, offering a convenient solution to a
number of analytical problems, such as resolution of multi component system, removal of
sample turbidity, matrix background and enhancement of spectral details. (C.H Vanselow
? To prepare a nickel sulphate complex or buffer.
? To estimate the total sugars using phenol sulphuric method.
? To determine phenol contents in plants using UV/Vis spectrometry.
? To determine the absorbance and apply Lamberts’ Beer’s Law using a UV/Vis
? To construct calibration curve based on Beer’s Law.
AIM: To determine an unknown concentration of nickel sulphate, sugars and plants.
MATERIALS AND METHODS
Quantitative analysis of NICKEL SULPHATE, SUGARS and PLANTS
Instrument: CARY UV/Vis spectrophotometer
Materials: cuvettes, test tubes, pipettes, concentrated sulphuric acid, dH2O
Reagent: nickel sulphate, phenol (5% w/v) in dH2O, Folin-Ciocalteau phenol
Software: CARY Win UV
Procedure 1 (practical 1)
The given aqueous solutions of 0,300; 0,200; 0,150; 0,100 and 0,050 mol.dm?3 were used to
calculate required volumes of nickel sulphate, those volumes were transferred into test tubes
and were diluted with distilled water to make up 2 ml. Six cuvettes were filled with different
concentration and one was filled with distilled water (blank). The absorbance was measured,
and maximum wavelength absorption was determined. The values were used to obtain the
absorbance as well as the unknown concentration.
Procedure 2 (practical 2)
Sugar solution was already prepared, different standard sugar solutions were prepared with
different concentrations of 10, 20, 30, 40, 50, 100?g/ml and the blank. Sugar solutions were
transferred into 6 test tubes and diluted with distilled water to make up to 2 ml, blank was
filled with distilled water only. 1 ml of phenol reagent was added to each test tubes, followed
by the addition of 5ml of concentrated sulphuric acid and was mixed probably. It was placed
for 30 minutes in the fume cardboard. After 30 minutes the solutions was transferred in
cuvettes and were placed in CARY UV/Vis spectrophotometer, different absorbance was
Procedure 3 (practical 3)
50?l of filtrate (plant sample) was added to the test tube along with 6950?l distilled water.
The test tube was swirled in order for the contents to mix. 0.5 ml of Folin-Ciocalteau phenol
reagent was added and test tube was swirled again to mix content again, and after 1 minute
but before 8 minutes, 1.5 ml of sodium carbonate solution (20g/100 ml) was added and mixed
again. Distilled water was added to make up volume of 10 ml. The stopper was placed on the
flask and carefully mixed (upturned) several times. It was placed in the fume cardboard for 30
minutes. Then after the solution was transferred to cuvette, and was place in CARY UV/Vis
spectrophotometer to observe the absorbance.
200, 100, 50, 20, 10?l of standard solutions (Gallic acid) were transferred into test tubes and
were diluted with a distilled water that made up to 1 ml, along with 1 ml of 40% ethanol was
added to each test tube and 6 ml of distilled water was added as well. Each test tubes were
swirled to mix the contents. 0.5 ml of Folin-Ciocalteau phenol reagent was added and test
tube was swirled again to mix content again, and after 1 minute but before 8 minutes, 1.5 ml
of sodium carbonate solution (20g/100 ml) was added and mixed again. Distilled water was
added to make up volume of 10 ml as well. The stopper was placed on the flask and carefully
mixed (upturned) several times. It was placed in the fume cardboard for 30 minutes. While
the solutions were in the fume cardboard, for a blank; 1 ml of 40% ethanol was transferred
into a test tube along with 6 ml of distilled water plus 0.5 ml of Folin-Ciocalteau phenol
reagent was added, 1.5 ml of sodium carbonate solution (20g/100 ml) was added along with
1 ml of distilled water to make 10 ml volume of solution and was mixed carefully.Then after
the solutions was transferred to six cuvettes including blank, and were place in CARY UV/Vis
spectrophotometer to observe the absorbance.
Note: all the solutions were placed in fume cardboard at the same time for 30 minutes
CALCULATIONS OF THE UNKNOWN
Quantitative analysis of Nickel Sulphate
STANDARD CONCENTRATION g/L ABSORBANCE
Std 1 0.050 0.3390
Std 2 0.100 0.6009
Std 3 0.150 0.8512
Std 4 0200 1.1184
Std 5 0.300 1.5434
CALCULATION OF THE UNKNOWN
m = ;#3627408460;2?;#3627408460;1
x = 0.1850 g/L
Estimation of total sugars by phenol sulphuric acid method
STANDARD CONCENTRATION (g/L) READINGS
Std 1 10 0.2332
Std 2 20 0.3704
Std 3 30 0.5127
Std 4 40 0.6552
Std 5 50 0.8190
CALCULATIONS OF UNKNOWN
= 0.0164 g/L
x = 90.70 g/L
x = 12.1037 g/L
x = 8.8232 g/L
Quantitative analysis of total phenol content in plants
Table 3: standard solution of gallic acid
STANDARD CONCENTRATION (g/L) READINGS
Std 1 10 0.5428
Std 2 20 0.2540
Std 3 50 0.5011
Std 4 100 0.8974
Std 5 200 1.6602
CALCULATION OF UNKNOWN
x = 214.2770 g/L
Absorbance of a sample is directly influenced by the concentration, the highly concentrated
solution absorbs more light and has a high refractive index (Jones, 2017). Several solutions
were prepared with different volumes of nickel sulphate and filled to a volume of 2ml. due
to different volumes the concentrations were also different. The table under caption
Practical 1 summarizes the results obtained when the samples put under
According to the table and graph plotted the absorbance shows linear and directly
proportional relationship. The samples with small volumes of the solutes or less
concentrated could absorb less light and transmit more. Which means the percentage of
transmittance is high in less concentrated solutions. The highly concentrated solutions have
particles that block the light from passing the solution and therefore more light don’t reach
to the detector. In such solutions the percentage of transmittance is very low and this is
seen in STD 4 and STD 5 in table 1.
A study conducted by Johann Heinrich Lambert who discovered the relationship between
these two factors stated a law “Beer-Lambert Law”. Its state that the quantity of light
absorbed by the substance dissolved in a fully transmitting solvent is directly proportional to
the concentration of the substance and the path length of the light through the solution.
The results show that the principle is observed since the proportionality is detected. (Jon H.
Phenol sulfuric method is the most commonly used method for determination of
carbohydrate concentration in many solutions. In the experiment the phenol-sulfuric acid
method of DuBois, Giles, Hamilton, Rebers and Smith which they developed in 1956 to
determine concentration and carbon content of carbohydrates in solution was used to test
the standard solution which was composed of galactose sugar. This method can be modified
with the use of UV spectroscopy.
The spectrometry results obtained in the experiment indicated in Table 2, shows a trend
between the concentration of standard and the light absorbance. Similar results were
reported by Ammar A. Albalasmeh, Asmeret Asefaw Berhe, Teamrat A. Ghezzehei (2013).
The graph of results above shows the absorbed light (y-axis) and concentration (x-axis) at
wavelength of 490nm. The relationship between the two appears to be directly proportional
because as the concentration was increased the amount of light absorbed also increased.
The principle behind the results is that when the sulfuric acid is added to the standard it
reacts with the sugar in the solution resulting in the hydrolysis of sugars to release the
furfurals that react with phenol to produce a colored compound which can absorb light and
give a certain absorbance reading. The unknown sample of sugar sample was calculated
using the results of the standard curve and those in table 2. According to Ammar A.
Albalasmeh et. Al (2013) the phenol- sulfuric acid method requires some time for color
development but when compared, the modified phenol-sulfuric acid UV method which uses
UV light absorption takes only a few seconds to complete. As proposed by DuBois et al
(1956) the phenol-sulfuric acid method requires approximately 30 minutes for phenol to
completely react with the furfurals and become stable to be read in the spectrophotometry.
The UV absorption of the furfurals in the modified method reaches a stable state within a
short time or as soon as the phenol and sulfuric acid has reacted with the solution. (Ammar
Albalasmeh A. et. al. (2013)).
The sulfuric acid needs to be concentrated to exert its dehydrating effect on sugar and since
sulfuric acid is corrosive the reaction should only be conducted under surveillance by a
Gallic acid is commonly used in pharmaceutical industry as a standard for determining phenol
content of various analyst for Folin-Ciocalteau. Gallic acid is also used as a synthetic
intermediate for production of pyrogallel and Gallic acid (Haines, 2002). Gallic acid was used
as standard solution and the total phenols were expressed as mg/ml gallic acid. Table 3
shows the variation mean the absorbance of Gallic acid. The wavelength of the maximum
absorbance was 760 nm. The absorbance increases as the concentration of the solutions
increases, because the darker the solution the more light is absorbed by the solution
The concentration of an absorbance was calculated using linear relationship between
absorbance and concentration, namely the Beer’s law. The solutions were prepared in the
range of concentration, where the unknown concentration was 0.00823, their
absorbance measured at a wavelength of 760 nm and absorbance was observed and
finally, a calibration function is deployed. The performance was checked within the range.
The sample (plant material) showed presence of phenolics. The content was determined
using Folin-Ciocalteau phenol reagent in terms of gallic acid equivalence and sample was
found to be 214.2770 g/L, At the maximum wavelength of 760 nm.
The results were not quite expected the data was askew due to experimental error, this
occurred when not transferring correct amount of concentration in one of the test tube,
throwing off the absorbance and the calibrate curve. Other error may be the solution
might have been expose to light since the Folin-Ciocalteau phenol reagent is sensitivity to
light and air because it might change colour and completely change the reaction.
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